The Her2 oncogene encodes a membrane-associated glycoprotein referred to as p185HER-2 having tyrosine kinase activity. Her2 is a member of the epidermal growth factor (EGF) receptor subfamily, which includes EGF receptor and Her3 and Her4 receptors (Kraus et al. Proc. Natl. Acad. Sci. USA 86, 9193-9197 (1989); Plowman et al. Proc. Natl. Acad. Sci. USA 90, 1746-1750 (1993)). The Her2 sequence was reported by Semba et al. (Proc. Natl. Acad. Sci. USA 82, 6497-6501 (1985)); Coussens et al. (Science 230, 1132-1139 (1985)) and King et al. (Science 229, 974-976 (1985)). A related rat gene was reported by Schecter et al (Nature 312, 515-516 (1984)).
Increased expression of the Her2 oncogene in tumor cells and cell lines has been reported by several groups (Coussens et al., supra; King et al., supra). The increased expression of Her2 results from gene amplification or increased expression of the single copy gene. These observations suggested that Her2 may be overexpressed in human cancer tissue. Slamon and colleagues (Slamon et al. Science 235, 177-182 (1987); Slamon et al. Science 244, 707-712 (1989)) examined Her2 expression levels in tumors taken from a large sample of breast and ovarian cancer patients. It was found that nearly 30% of those patients had amplification and over-expression of the Her2 gene which was associated with a poor clinical outcome (increased relapse and low survival rate) particularly in node-positive breast cancer patients. The correlations reported by Slamon have been confirmed in a number of studies (see, for example, Ro et al. Cancer Res. 49, 6941-6944 (1989); Walker et al. Brit. J. Cancer 60, 426-429 (1989); Wright et al. Cancer Res. 19, 2087-2090 (1989); Berchuck et al. Cancer Res 50, 4087-4091 (1990); Kallioniemi et al. Int. J. Cancer 49, 650-655 (1991); Rilke et al. Int. J. Cancer 49, 44-49 (1991)).
The presence of certain factors, such as Her2 overexpression, that are indicative of a poor prognosis may suggest that adjuvant therapy after surgical removal of the tumor is appropriate. Adjuvant therapy can include high dose chemotherapy and autologous bone marrow transplantation. It has recently been reported (Muss et al. N. Engl. J. Med. 330, 1260-1266 (1994)) that breast cancer patients having tumors displaying Her2 overexpression enjoyed significant benefits from adjuvant therapy.
By analogy with other receptor protein tyrosine kinases, it is assumed that a ligand for Her2 stimulates receptor phosphorylation. A number of polypeptide factors have been reported to increase tyrosine phosphorylation of Her2 and were presumed to be a ligand (Wen et al. Cell 64, 559-572 (1992); Holmes et al. Science 256, 1205-1210; Marchionni et al. Nature 362, 312-318 (1993); Falls et al. Cell 72, 801-815 (1993)). However, there is no evidence that any of these factors are true ligands which bind directly to Her2 and stimulate receptor phosphorylation. One approach to circumvent the absence of ligand is to generate a ligand-like monoclonal antibody (mAb). Several groups have generated anti-Her2 mAbs using either a cell-surface Her2 receptor or a purified extracellular domain of Her2 receptor (Yarden, Proc. Natl. Acad. Sci. USA 87, 2569-2573 (1990); Hanwerth et al. Br. J. Cancer 68, 1140-1145 (1993); Srinivas et al. Cancer Immunol. Immunother. 36, 397-402 (1993); Stancovaski et al. Proc. Natl. Acad. Sci. USA 88, 8691-8695 (1991)). These mAbs stimulated tyrosine phosphorylation of Her2 from overexpressing cells, but were not fully characterized in terms of binding to and phosphorylation of each of Her2, Her3 or Her4 or in terms of the kinase activation in Her2 transfected cells.
Growth inhibitory effects of anti-Her2 mAbs on breast cancer cells have been reported previously (Tagliabue et al. Int. J. Cancer 47, 933-937 (1991); Hudziak et al. Mol. Cell. Biol. 2, 1165-1172 (1989); Drevin et al. Oncogene 2, 387-394 (1988); Fendly et al. Cancer Res. 50, 1550-1558 (1990); Hanwerth et al., supra; see also review by Vitetta and Uhr, Cancer Res. 54, 5301-5309 (1994)), but these effects were interpreted to be cytostatic since removal of antibody allowed resumption of cell growth. Xu et al. (Int. J. Cancer 53, 401-408 (1993)) reported anti-Her2 antibodies which were cytotoxic for anchorage-independent tumor cell growth.
An anti-EGF receptor mAb was reported to induce apoptosis on the human colorectal carcinoma cell line, DiFi, which overexpresses EGF receptor, and to induce morphological changes at concentrations of 5 to 20 nM. These effects were interpreted in terms of both blockage of EGF binding to the cognate receptor by the competing mAb and lack of the mAb mitogenic activity (Wu et al. J. Clin. Invest. 95, 1897-1905 (1995)).
Apoptosis, or programmed cell death, is a form of cell death characterized by cell shrinkage and DNA fragmentation. Collapes of the cell nucleus is apparent as chromation is fragmented into single or multiple mononucleosomal units, a process mediated by an endogenous endonuclease. Apoptosis is distinct from necrotic cell death which results in cell swelling and release of intracellular components (Kerr et al. Br. J. Cancer 26, 239-257 (1972); Wyllie et al. Int. Rev. Cytol. 68, 251-306 (1980); Wyllie Nature 284, 555-556 (1980)). Apoptotic cells, without releasing such components, are phagocytosed and hence degraded (Savill et al. Nature 343, 170-173 (1990)). Therefore, apoptosis results in an efficient process for elimination of nonviable cells by the host's own defense mechanisms.
It is an object of the invention to generate antibodies to Her2 which induce apoptosis in Her2 expressing cells and thereby “tag” such cells for removal from the host. The antibodies are useful for inducing apoptosis in tumors. This represents a substantial improvement over currently available antibody therapy for cancer which typically involves killing tumor cells by antibody in conjuction with a cytotoxic agent. Cytotoxic agents generally produce undesirable side effects which, if severe, can lead to a reduction or interruption of treatment. The present approach allows for killing of tumor cells by the host immune system, thereby avoiding the effects of cytotoxic agents and tumor cell necrosis induced by such agents.